Processes for in vivo production of astaxanthin and phaffia rhodozyma yeast of enhanced astaxanthin content

ABSTRACT

An economical process for in vivo production of the pigment astaxanthin, and particularly a process for enhancing astaxanthin content of cultures of microorganisms of genus  Phaffia , the process comprising culturing a microorganism of genus  Phaffia  in a nutrient medium containing an antibiotic, a cytochrome B inhibitor, or a terpenoid synthetic pathway inhibitor, cultivating surviving pigment enhanced microorganisms, and harvesting the yeast.

This is a Continuation of application Ser. No. 08/067,797 filed 27 May1993, now abandoned, which is a continuation of U.S. Ser. No. 07/837,120filed 14 Feb. 1992, now U.S. Pat. No. 5,356,809, which is a divisionalof U.S. Ser. No. 07/399,183 filed 23 Aug. 1989, now U.S. Pat. No.5,182,208, which is a continuation of U.S. Ser. No. 07/385,961 filed 28Jul. 1989, now abandoned, which is a continuation-in-part of U.S. Ser.No. 07/229,536 filed 8 Aug. 1988, now abandoned.

FIELD OF THE INVENTION

The invention pertains in one aspect to an economical process for invivo production of the pigment astaxanthin. In another aspect, theinvention pertains to a process for enhancing astaxanthin content ofcultures of microorganisms of genus Phaffia, the process comprisingculturing a microorganism of genus Phaffia in a nutrient mediumcontaining an antibiotic, a cytochrome B inhibitor, or a terpenoidsynthetic pathway inhibitor, cultivating surviving pigment enhancedmicroorganisms, and harvesting the yeast.

BACKGROUND OF THE INVENTION

The reddish carotenoid pigment astaxanthin is commonly found in natureand is conspicuously displayed by a number of animals. Animals unable tosynthesize this pigment rely on dietary intake of this pigment or apigment precursor.

The red skin and flesh color of naturally occurring salmon and trout isdue primarily to astaxanthin, which is usually present as an unboundpigment in these fishes. In nature, marine zooplankton and nekton in thediet provide salmon with their carotenoid pigments.

Due to a lack of dietary astaxanthin, fish raised on fish-farms or inhatcheries are generally pale and lack the skin and flesh colorscharacteristic of fish grown in their natural environment. Whether ornot the carotenoids are nutritionally important in the animal or humandiet has not been determined, but pigments do make certain foodsattractive. That is, since the color of a food is frequently anindicator of its quality, there is a strong consumer preference for fishhaving natural coloration, even though nutritionally the pale farmproduced fish may be identical to those grown in their naturalenvironment. There is also evidence that astaxanthin or its precursorcontributes to the distinctive flavor of baked salmon.

Recent increasing concern over health risks has resulted in a ban onvarious synthetic coloring agents which have a potential ofcarcinogenicity and/or teratogenicity. Yellow and red azo dyes which areincreasingly prohibited from use in foods are being replaced withnon-toxic carotenoids. The carotenoids are generally not toxic even athigh levels. Thus, naturally occurring carotenoids are the preferredpigment for coloring, e.g., salmonids.

In the past, numerous studies have been carried out utilizing carotenoidcontaining crustaceans or crustacean processing wastes in salmoniddiets. The pale color of fish produced on fish-farms or in hatcheries isimproved when the fish are fed a diet supplemented by large quantitiesof dried, ground-up exoskeletal crustacean remains. Crustacean shellsare, however, very low in carotenoid content and high in minerals which,without extensive processing to improve their dietary quality, restrictstheir inclusion in salmonid diets. Further, a satisfactory color can bedeveloped in this manner of feeding only over long periods of time andit is desirable, if not indeed essential in the economic sense, todevelop satisfactory colors within very short periods of time.

It is known that astaxanthin per se can be added to fish food to improvefish color. For example, U.S. Pat. No. 4,239,782 describes a method forenhancing the color of fish comprising adding to the fish food apigmenting agent such as astaxanthin and additionally limited amounts ofthe hormone testosterone, which hormone acts as a catalyst incombination with a chosen pigmenting agent or agents to enhance thecolor of fish.

The two primary commercial sources for astaxanthin per se are extractsfrom crustacean shells and chemical synthetics. The red carotenoidpigment can be extracted from the exoskeletal crustacean shells andtissues and fed, admixed with other feed in dietary formulations, to thefarm fish, crustacea and certain fowls in massive concentrations todevelop satisfactory skin, flesh, carapace or egg yolk pigmentations.Examples of processes for extraction of astaxanthin from crustaceanshell and tissue waste are described, for example, in U.S. Pat. Nos.3,906,112 (Anderson) and 4,505,936 (Meyers et al).

In an article in Journal of Food Science, Volume 47 (1982), entitled“Extraction of Astaxanthin Pigment from Crawfish Waste Using a Soy OilProcess”, various extraction techniques are described. For example,whole crawfish waste is ground-up, the comminuted crawfish waste isadmixed with water, the pH is adjusted with an alkali or acid, an enzymeis added to the solution, and the solution stirred, heated andhydrolyzed. After hydrolysis, the astaxanthin is extracted with oil andthe astaxanthin enriched oil recovered by centrifugation. However, thecost of natural isolates of astaxanthin, especially from krill andcrawfish shells, can cost anywhere from $5,000 to $15,000 per kilogram.Obviously, a less source dependent and more economical process forproduction of astaxanthin is needed.

Pigmentation of salmon and trout flesh has also been accomplished usingthe synthetic carotenoid canthaxanthin as a feed additive, but thischemical is rather expensive and has been reported to produce a somewhatunsatisfactory color in salmonids. Recent work in chemical synthesis ofastaxanthin is exemplified by U.S. Pat. Nos. 4,245,109 (Mayer et al),4,283,559 (Broger et al), and 4,585,885 (Bernhard et al). The presentcost of synthetic astaxanthin pigment is approximately $2,000 perkilogram. Many countries, however, prohibit the use of syntheticcarotenoids.

Astaxanthin remains one of the most expensive ingredients used in salmonfeed for pen-reared salmon. As the interest in aquaculture, i.e.,farming fish, has exploded recently, the commercial demand for aneconomical source of astaxanthin has grown proportionately.

The pigmentation of avian egg yolks has also been studied because of theeconomic importance of color in chicken egg yolks. Yolks with a highpigment content are demanded. The most common pigment source incommercial diets has been yellow corn, which supplies the prominent eggyolk pigments cryptoxanthin, zeaxanthin and lutein. Unfortunately,higher energy grains such as milo, wheat, rice and barley are replacingcorn in the chicken diet, with the consequent loss in pigmentation.Astaxanthin can be used as a poultry food supplement to increase yolkpigmentation.

One approach not presently commercially employed in production ofastaxanthin is biosynthesis, i.e., employment of microorganisms tosynthesize astaxanthin.

As a microbial source of astaxanthin, the yeast Phaffia rhodozyma isknown (Johnson, “Astaxanthin Production by the Yeast Phaffia rhodozymaand its use as a Pigment Source in Animal Feeding”, Masters Thesis,University of California at Davis, 1976). Yeast is generally consideredto be a highly nutritious feedstuff and is often desirable in animaldiets; the additional attribute of containing astaxanthin suggests thatP. rhodozyma may be an ideal feed supplement for animals that require adietary source of this pigment, e.g. salmonids, crustaceans, layinghens, or birds such as flamingoes.

However, the pigment yield of naturally occurring P. rhodozyma is onlyin the order of 200 to 600 ppm/dry weight of yeast in 6 day growth. As asource of astaxanthin per se, naturally occurring P. rhodozyma isinadequate. Experiments in supplementing the diets of certain fish,crustaceans and fowl with naturally occurring P. rhodozyma showed somepromise. Practically, however, the large volume of P. rhodozyma whichmust be added as a food supplement in order to obtain satisfactorylevels of pigmentation detracts from the commercial suitability ofnaturally occurring P. rhodozyma as a pigment source.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provideeconomical process for in vivo production of the pigment astaxanthin.

Another object of the present invention is to provide a process forobtaining cultures of the yeast Phaffia rhodozyma having increasedastaxanthin content. Another object of the present invention is toprovide P. rhodozyma characterized by a high astaxanthin content.

It is another object of the present invention to develop a process forimproving astaxanthin content of progeny derived from naturallyoccurring P. rhodozyma such as strain ATCC-24230 or ATCC-24202 depositedwith the ATCC.

Yet another object of the present invention is to develop a process forimproving astaxanthin content of mutated strains of the yeast P.rhodozyma.

These and other objects have been attained by a process comprising, inits most basic form, culturing a microorganism of genus Phaffia in anutrient medium containing an antibiotic, a cytochrome B inhibitor, or aterpenoid synthetic pathway inhibitor, cultivating surviving pigmentenhanced microorganisms, and harvesting the yeast.

The key step in strain development is the morphological selection step.There is no limit to the possible combinations of sequence and type ofmutation and antibiotic, a cytochrome B inhibitor, or terpenoidinhibitor selection to which P. rhodozyma may be subject within thepresent invention. Recent results confirm the reproducibility of thistechnique.

Related objects and advantages of the present invention will become moreapparent by reference to the following figures and detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The process and apparatus in accordance with the present invention willbe described with reference to the accompanying drawings, in which:

FIG. 1 shows the chemical structure and relationship of various pigmentsand intermediates found in P. rhodozyma.

FIG. 2 is a flow chart showing preferred sequences of mutant isolationsin P. rhodozyma.

FIG. 3 is a micropictograph showing the appearance of variegatedcolonies.

DETAILED DESCRIPTION OF THE INVENTION

During an expedition in 1967 for the purpose of studying yeast floraeassociated with trees on the major Japanese Islands and in the PacificNorthwest, a peculiar astaxanthin synthesizing organism was isolatedfrom slime fluxes of various broad-leafed trees (Phaff et al., “Acomparative study of the yeast florae associated with trees on theJapanese Islands and on the west coast of North America”, Proc. IV:Ferment. Technol. Today, (1972) pp. 759-774). This organism, now knownas Phaffia rhodozyma, was determined to have the capabilities ofproducing carotenoid pigments and fermenting several sugars.

Phaffia has been granted a genus because of its unique characteristics,which include its ability to ferment glucose and other sugars (comparedto the other carotenoid-forming yeasts which are all strictly aerobic),its synthesis of astaxanthin as its principal carotenoid, its mode ofbud formation, its possession of certain metabolic properties such asthe ability to use urea which is less common in ascomycetous yeasts, andits cell wall composition. P. rhodozyma, the only species in this genus,reproduces exclusively by budding, and an examination of this mode ofvegetative reproduction by scanning electron microscopy reveals that theyeast buds repeatedly from the same site which leads to the formation ofthe multilayered cell wall.

Other characteristics of Phaffia include its formation ofchlamydospores, its mol % G+C content of 48.3%, its ability to spliturea by urease, and its apparent lack of a perfect stage. Its propertiessuggest that the yeast is of basidiomycetous affinity. Attempts to findthe sexual cycle of Phaffia have all failed.

Andrewes et al, as reported in “Carotenoids of Phaffia rhodozyma, a redpigmented fermenting yeast”, Phytochem. 15, (1976) pp. 1003-1007, foundthe carotenoids of P. rhodozyma to be unusual. Pigments and pigmentintermediates found by Andrewes in naturally occurring P. rhodozyma areshown in FIG. 1 where (A) is echinenone, (B) is 3-hydroxyechinenone, (C)is phenicoxanthin, and (D) is astaxanthin. Astaxanthin was found to bethe major pigment synthesized by this yeast; in the naturally occurringyeast it comprised approximately 85% of the carotenoid mixture.

While the carotenoids of most plant and animal sources can be readilyextracted with water-miscible solvents, the yeasts are well known forthe tenacious attachment of their pigments. Astaxanthin is firmlyattached in the yeast cell and not extractable by lipid solvents unlessthe structure of the yeast cell is first altered.

The most quantitative method of extraction involves mechanicallybreaking the yeast cells such as in a French press (Simpson et al.,“Modified French press for the disruption of microorganisms”, J. Bact.,86, (1963) pp. 1126-1127) or in a Braun homogenizer (Bae et al., “Theoccurrence of plectaniaxanthin in Cryptococcus laurentii”, Phytochem.,10, (1970) pp. 625-629), and then extracting the cells with solvents.These methods are used routinely for estimation of astaxanthinconcentration.

The need for the large scale recovery of astaxanthin led to thedevelopment of an enzymatic method of extraction. The method utilizedextracellular lytic enzymes produced by the bacterium Bacillus circulansWL-12, which partially digested the yeast cell wall and rendered thecarotenoid pigments extractable by lipid solvents. Complete extractionof astaxanthin from heat-killed P. rhodozyma cells was obtained aftergrowing B. circulans WL-12 on these yeast cells for 26 hours and thenextracting the yeast-bacterium mixture with acetone.

A bacteria-free lytic system, which gave quantitative extraction ofastaxanthin from P. rhodozyma, was obtained by concentrating the culturebroth from the growth of B. circulans WL-12 on P. rhodozyma cells.Preferably, B. circulans WL-12 is grown on a medium containing P.rhodozyma cells. The lytic system was found to work most efficiently atpH 6.5 and with low concentrations of yeast.

Generally, nutrition and environment have an effect on carotenoid yieldin various carotenoid producing microorganisms. Enhancement ofcarotenoid formation in fermentations in complex media has been reviewed(Hanson, “Microbial production of pigments and vitamins”, in MicrobialTechnology, edited by H. J. Peppier, Reinhold, New York, (1967) pp.222-250).

Carotenoids are tetraterpenes and their basic pathway of synthesis issimilar to that of other terpenoids. Acetyl CoA is the initial precursorand the first specific terpenoid compound is mevalonic acid (Schopfer etal, “Sur la biosynthése du β-caroténe par Phycomves cultivé sur un mileucontenant de l'acétate de sodium comme unique source de carbone”,Experientia, 8, (1952) p. 140). Isopentenyl pyrophosphate is thefundamental precursor from which carotenoids are derived (see Goodwin,“Biosynthesis”, Carotenoids, Edited by O. Isler, Basel, Birkhauser,(1971) pp. 577-636; Britton, “Biosynthesis of Carotenoids”, in Chemistryand biochemistry of plant pigments, Vol. 1, (1976) pp. 262-327).

Most previous work with P. rhodozyma has been done with the type strain#67-210. Visual examination of young colonies of several additionalnatural isolates (UCD-FST #s 67-202, 67-203, 67-210, 67-383, 67-385 and68-653C) indicated that 67-210 and 67-385 (American Type CultureCollection ATCC Nos. 24202 and 24230 respectively) were the most highlypigmented strains. Quantitative determination of astaxanthin content inthese two strains after 5 days growth in YM broth indicated that 67-385contained ≈450 μg/g dry yeast in comparison with ≈295 μg/g in 67-210.67-385 was primarily employed by the inventors for strain developmentbecause of its higher natural astaxanthin content.

As a result of a series of experiments designed to determine the optimalculture conditions for growth and pigmentation of P. rhodozyma, it wasfound that astaxanthin biosynthesis occurred maximally during theexponential phase of growth. The pigment yield in the growth medium wasfound not to be solely dependent on cell concentration but wasinfluenced by the culture conditions. The optimal pH for astaxanthinproduction was found to be 5.0 in shake flasks. At the other pHs tested,however, the concentration of astaxanthin in P. rhodozyma remainedrelatively constant.

The temperature of cultivation was found to influence the growth rate ofP. rhodozyma but not the accumulation of astaxanthin in the yeast cell.The effects of light also did not affect carotenogenesis in P.rhodozyma, although the cells grown under high light intensity appearedto have a redder hue, which may have been due to differentconcentrations of particular carotenoids.

It was found that a low glucose concentration and high air supplypromoted efficient astaxanthin production by P. rhodozyma. Theconcentration of astaxanthin in the red yeast decreased considerably ifthe air supply was below 20 mmoles/h or the glucose concentration wasabove 4% w/v. However, astaxanthin was still the predominant pigment inyeast cultured under either of these conditions. If the effects of lowair and high glucose were combined, however, then the astaxanthinconcentration in P. rhodozyma was decreased to extremely low levels andthe formation of B-zeacarotene occurred. Under these adverse conditions,again, astaxanthin was not efficiently formed from the carotenes.

When cultured on carbon compounds which repressed ethanol production inthis yeast (e.g. cellobiose) the yields of astaxanthin werecomparatively high. If cultured on carbon sources which presumablypromoted ethanol production (e.g. high glucose) the astaxanthin yieldswere comparatively low. Carbon compounds metabolized via thepentosephosphate pathway (e.g. xylose) did not promote efficientcarotenogenesis.

Despite extensive experimenting in optimizations of nutrient medium andenvironmental conditions on naturally occurring strains of P. rhodozyma,a dramatic increase in pigment content was not attained. The inventorsconcluded that manipulation of the above discussed factors alone may notbe sufficient to induce increase in astaxanthin content of P. rhodozymato the extent necessary to render biosynthesis commercially feasible.

With the objective of isolating a novel genetic mutant of P. rhodozymacapable of increased astaxanthin production, P. rhodozyma was subject tomutagenesis. However, results were inconsistent, presumably due to therandom and non-directed nature of mutation. For example, attempts atisolating highly pigmented variants were not successful after screeningseveral thousand colonies after UV exposure. Most of the UV-generatedmutants were considerably reduced in astaxanthin content and were verypale.

Potential for further progress in improvement in astaxanthin content inP. rhodozyma by conventional mutagenic techniques appears to be limited.This is because microorganisms possess tight genetic regulatory controlsover the biosynthesis of their cellular components so that they tend notto overproduce unnecessary cellular constituents. Since the biosynthesisof pigment such as astaxanthin is apparently tightly regulated in anygiven strain of yeast cells, the chances of producing a viableinheritable genetic alteration to produce colonies capable of increasedastaxanthin production by undirected mutagenesis is probably very low.

Since screening of colonies after mutagenesis was relativelyunsuccessful, the inventors tried to develop selection procedures forastaxanthin overproduction. Because astaxanthin formation is decreasedby high concentrations of glucose, the inventors tried 2-deoxyglucose asa selective agent to isolate glucose-resistant strains that might becatabolite-derepressed and, in turn, exhibit enhanced pigmentbiosynthesis. Although some strains generated by this selectiveprocedure were changed in pigmentation, many were extremely unstable andnone were increased in astaxanthin concentration more than two-fold overthe naturally occurring parent.

Inhibitors of sterol biosynthesis including ketoconazole and miconazolewere incorporated into yeast malt extract medium agar, which resulted insignificant killing of P. rhodozyma. Screening of several thousandsurvivors, however, did not produce highly pigmented yeast strains.Several other compounds tested including nicotine (1 mM), imidazole (4mM), 2-methylimidazole (1 mM), and morpholine (10 mM) did visuallychange the color of the colonies, but did not impair growth, whichindicated a change in carotenoid composition. However, since thesevariants were not increased in astaxanthin, no further analysis in thechanges in pigment compositions was carried out.

The inventors also tried treating P. rhodozyma with several inhibitorsof electron transport including thenoyltrifluoroacetone (TTFA),antimycin A, and sodium cyanide. Naturally occurring P. rhodozyma wassubstantially killed by low concentrations of antimycin A and TTFA, butwas found to be relatively insensitive to cyanide and azide.

As a result of extensive experimentation, discussed above in part, theinventors made a surprising discovery of a process by which colonies ofP. rhodozyma can be produced, which colonies are characterized by a highastaxanthin content. P. rhodozyma produced by the process of the presentinvention is non-reverting, the astaxanthin is present in the yeast in asufficiently high concentration to be a more effective dietary food andpigment supplement than naturally occurring P. rhodozyma, and coloniesof the yeast can be obtained which are able to produce astaxanthin insufficient quantities to render astaxanthin fermentation a commerciallyfeasible process for production of astaxanthin.

More specifically, the inventors have discovered that by subjecting anaturally occurring or mutated strain of P. rhodozyma to growth in thepresence of a metabolic pathway inhibitor, particularly a mainrespiratory pathway inhibitor, in the presence of an influence such asan agent or environmental condition which triggers a secondaryrespiratory pathway, or morphological selection using a selecting agent,and particularly an antibiotic, a cytochrome B inhibitor, or a terpenoidsynthetic pathway inhibitor as a selecting agent, a directed andspecific selection of highly pigmented P. rhodazyma takes place. Forexample, plating of the pink yeast, P. rhodozyma, to agar containing anantibiotic, a cytochrome B inhibitor, or a terpenoid synthetic pathwayinhibitor gave rise to colonies of unusual morphology characterized by anonpigmented lower smooth surface that developed highly pigmentedvertical papillae after 1 to 2 months. Isolation and purification of thepapillae, followed by testing for pigmentation in shake flasks,demonstrated that several mutans were increased 3 to 6 fold inastaxanthin content compared to the parental natural isolate.

One of the selected progeny (IGI-887J0; see FIG. 2 and accompanyingtext) was characterized physiologically. The mutant grew slower onvarious nitrogen sources and had lower cell yields on several carbonsources. It showed increased sensitivities to the respiratory inhibitorssuch as antimycin A and TTFA, but did not differ from the naturalisolate in sensitivity to cyanide or azide. It was more sensitive tokilling by hydrogen peroxide. Analysis of the carotenoids by thin layerchromatography showed two unknown carotenoids not present in the parent,as well as an increased accumulation of carotenes and cis-astaxanthin.

Antibiotic selection agents include, for example, one or more ofantimycin, tunicamycin and nystatin. Antimycin may also be classified asan electron transport inhibitor or a cytochrome B inhibitor. Othercytochrome B inhibitors which enhance pigmentation of select progenyinclude, for example, 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HOQNO).The terpenoid synthetic pathway inhibitors include, for example,mevalonic acid lactone, which may be generally referred to as ametabolic inhibitor, and which is an example of a sterol inhibitor.

Concentrations of selection agents, e.g., antimycin, preferably rangefrom 1 to 100 μM on yeast malt extract medium (YM) plates, morepreferably 30 to 80 μM, and most preferably, for generation of the mostdistinctive colonies, are approximately 50 μM.

The effect of the antibiotic, cytochrome B inhibitor, or terpenoidsynthetic pathway inhibitor on P. rhodozyma is surprising and thespecific underlying mechanism has yet to be elucidated. It is surprisingthat P. rhodozyma subjected to antibiotic, cytochrome B inhibitor, orterpenoid synthetic pathway inhibitor selection produce colonies ofhigher pigment contents, while experiments conducted with other agents,such as respiratory inhibitors including KCN, rotenone, TTFA and others,do not seem to significantly influence pigmentation of P. rhodozyma onYM plates or in liquid growth media.

The inventors have developed a hypothesis which may provide a possibleexplanation for the effect of antimycin on P. rhodozyma, but thishypothesis is speculative and should not be taken to effect the scope ofthe invention.

The inventors suspect that since ring closing and hydroxylation maydepend on cytochrome P450 as occurs in sterol synthesis, and acytochrome B inhibitor such as antimycin reacts with cytochrome B whichis the same cytochrome that reduces cyt. P450, perhaps the increasedpigmentation in the antimycin mutants is due to altered cyt. P450function or activity. Also this molecule is known to inactivateglutamine synthetase and this may be the reason for an observed slowingof nitrogen catabolism. A further discussion of the hypothesis can befound in An et al, “Isolation of Mutants of Phaffia rhodozyma withincreased quantities of Astaxanthin”, unpublished manuscript, authoredin significant part by a co-inventor, a copy of which is providedherewith.

Recent results confirm that this technique can work repeatedly with P.rhodozyma.

The naturally occurring or antibiotic, cytochrome B inhibitor, orterpenoid synthetic pathway inhibitor selected yeast cells mayadditionally be exposed to a mutagen before, after, or before and afterselection, or in any desired number or combination of mutation andselection steps, so long as antibiotic, cytochrome B inhibitor, orterpenoid synthetic pathway inhibitor selection is included in theprotocol.

Mutagens or mutagenic agents can be selected from a variety of chemicalsor other exposures, and are preferably powerful enough to causeproduction of nonrevertable mutations. Mutants wherein no degenerationhas been observed are highly desirable for industrial uses. Strongmutagens include ethyl-methane sulfonate, nitrosoguanidine(N-methyl-N-nitro-N-nitroso-guanidine), nitrous acid (though relativelyhigh amounts may be needed), ultraviolet radiation in significantamounts, x-ray, and others.

Presently preferred are nitrosoguanidine and UV because of their readyavailability, their relatively powerful mutagenic nature, and theirrelative ease a . . . j safety of handling. However, any strong mutagencan be employed, using amounts and exposure techniques as known in theart.

In some cases a weak mutagen can be combined with a strong mutagen.Among the weak mutagens are 2-aminopurine, t-bromouracil, hydroxylamine,sodium bisulfite, and others.

The sequence of mutations and selections with which the highestastaxanthin yielding strains to date have been produced is shown in FIG.2. With reference to FIG. 2, the naturally occurring parent 67-385,obtained from the American Type Culture Collection ATCC No. 24230, wasassayed as containing 200-600 μg/g dry weight of yeast in 6 day growth.67-385 was subjected to antimycin selection, and higher pigmentedprogeny, referred to as 10′-887J0 were obtained. The first mutantpapillus dissected from the basal colony contained approx. 700 μg totalcarotenoid/g yeast compared to the parental strain which had 300 to 400μg/g. 10′-887J0 was assayed to have an astaxanthin content of 960 μg/gdry weight of yeast in 6 day growth. 10′-887J0 was replated to YM agarcontaining 50 μM antimycin and second generation antimycin colonies wereselected. One of these produced 1200 μg/g and another produced 1450μg/g. Therefore, it appeared that antimycin A is an excellent selectiveagent for isolating pigmented variants of P. rhodozyma.

IGI-887J0 were subject to nitrosoguanidine (NTG) mutation and IGI-887J2were obtained which produced 1200 to 1500 μg total carotenoids/g yeast.IGI-887J2 were further selected with antimycin to produce IGI-887J1which was assayed to have an astaxanthin content of 700-1100 μg/g dryweight of yeast in 6 day growth. A second NTG mutagenesis producedIGI-887J3 progeny.

Upon purification of colonies of IGI-887J3 the isolates segregated intowhite and pigmented colonies. The white revertant was designatedIGI-887J4. The pale colonies grew very poorly on ammonium sulfate at 5g/l, grew a little better on high levels of ammonium sulfate (20 g/l),and on plates grew well on glutamate or glutamine. These results suggestthat the strains are progressively impaired in nitrogen metabolism whichlimits growth rate.

IGI-1287J1 was isolated after nitrosoguanidine mutation of IGI-887J4,the white segregant of IGI-1887J3. IGI-1287J1 was assayed as containing900-1400 μg/g dry weight of yeast in 6 day growth. This strain also wasimpaired in nitrogen metabolism. It was noted that strains IGI-887J3 andIGI-1287J1 do not grown on ethanol, while the prior mutants do.

IGI-2880B60 was obtained from IGI-887J2 by NTG mutagenesis, and wasassayed to contain 1700 μg/g dry weight of yeast in 6 day growth.

Characterization of the Antimycin Mutants.

The parental strain (67-385), IGI-887J0, and IGI-887J2 had approximatelyequal yields of cells with ammonium, glutamate or glutamine as nitrogensources. However, analysis of growth rates showed that the series ofstrains grew progressively slower on these nitrogen compounds. Thesedata suggested that the rate or efficiency of nitrogen utilization wasimpaired in the antimycin mutants.

The parent and two studied mutants had reduced cell yields on fourcarbon sources tested, but were more highly pigmented. The antimycinmutants still fermented glucose and also had significant ethanoldehydrogenase activity. However, IGI-887J2 no longer grew on ethanol andhad reduced cell yields on other respiratory substrates includingsuccinate. The reduced yields on the energy sources and the specificityof inhibition by antimycin for the electron transport chain suggestedthat respiration or mitochondrial function in the pigmented mutants wasaltered.

Sensitivities of the mutants to various respiratory inhibitors includingantimycin A, theonyltrifluoroacetine, sodium azide, hydrogen peroxideand sodium cyanide were examined. These inhibitors affect differentregions of the respiratory chain. Surprisingly, the antimycin-inducedmutants were more sensitive to antimycin A on YM plates and also inliquid media. The parental strain had approximately 50% survival at 100μM antimycin, compared to the mutants which did not survive about 60 μM.The concentrations of antimycin that killed 50% of the populations of10′-887J0 and IGI-887J2 on YM agar were approximately 18 and 3 μM,respectively. The mutants were more sensitive in liquid media and werekilled at antimycin concentrations near 0.5 μM.

These results clearly show that the pigmented papillae that arose fromthe pale colonies were actually more sensitive to the drug even thoughthey were isolated on plates containing antimycin. Apparently, thespatial separation of the papillae from the agar facilitated thegeneration of the more sensitive strains.

The mutants were also quite sensitive to other respiratory inhibitorsincluding TTFA and hydrogen peroxide, but were only slightly moresensitive to cyanide, and did not differ in sensitivity to azide. Thesedata supported the above hypothesis that the antimycin-isolated strainspossessed an altered respiratory chain, and suggested that a lesion mayoccur in the early regions of the chain, near cytochrome b.

For carotenoid composition of mutants, see An et al, “Isolation ofMutants of Phaffia rhodozyma with increased quantities of Astaxanthin”,unpublished manuscript, authored in significant part by a co-inventor, acopy of which is provided herewith.

It is necessary to supply suitable amounts of nutrients and minerals inthe feed media in order to assure proper microorganism growth, tomaximize assimilation of the carbon energy source by the cells in themicrobial conversion process, and to achieve maximum cellular yieldswith maximum cell density in the fermentation media.

The composition of the ferment can vary over a wide range, depending inpart on the yeast strain, the substrate employed, and the mineralscontent in the ferment (that is, liquid plus cells). Set forth in thetable below are the minimum, broad, and presently preferred ranges ofconcentrations of various elements in the ferment, the concentrationbeing expressed as of the element, though it is recognized that all orpart of each can be present in the form of a soluble ion, or in casessuch as P, in a combined form of some type such as phosphate. The amountof each element is expressed in grams or milligrams per liter of ferment(aqueous phase, including cells):

Weight of Element per Liter of Ferment Element Minimum Broad RangePreferred Range P 0.2 g 0.2-5 g 0.4-2 g K 0.1 g 0.1-3 g 0.1.0.7 g Mg0.15 g 0.15-3 g 0.3-1.2 g Ca 0.06 g 0.06-1.6 g 0.08-0.8 g S 0.1 g 0.1-8g 0.2-5 g Fe 0.5 mg 0.5-30 mg 0.6-20 mg Zn 2 mg 2-100 mg 3-40 mg Cu 0.6mg 0.6-16 mg 1-10 mg Mn 0.6 mg 0.6-20 mg 0.9-8 mg

The manner in which to carry out the present invention, and furtherfeatures and advantages of the present invention, will be apparent fromthe following illustrative Examples. The Examples are provided for thepurpose of promoting an understanding of the principles of theinvention. Although the conditions and language in the examples isspecific, it will nevertheless be understood that no limitation of thescope of the invention is thereby intended, such alterations and furthermodifications and applications of the basic principles of the presentinvention as illustrated therein being contemplated as would normallyoccur to one of ordinary skilled in the art to which the inventionrelates.

In the Examples, naturally occurring strains are obtained from theofficial depository, the American Type Culture Collection in Rockville,Md., and given ATCC Nos. 24230 and 24202. The ATCC designations reflectthat two agar slant cultures of each strain have been deposited with theofficial depository.

Mutagenesis Example 1 Ultraviolet Mutagenesis

Strain IGI 2880860 was mutagenized with ultraviolet light from agermicidal UV lamp at a distance of 12 inches from a saline suspensionof cells for 1 min. (95.3% kill), 3 min. (99.4% kill), and 5 min (99.99%kill). Mutagenized cells were kept in the dark for 1 hr. following theperiod of mutagenesis and plated on yeast malt extract medium (YM) atseveral dilutions. Plates were incubated at 20° C. Dark red orangecolonies were isolated on the basis of observed pigmentation differencesand streaked to slants.

Example 2 Nitrosoguanidine Mutagenesis

Stain 1287J1 was grown in YM for 48 hr. Cells were pelleted bycentrifugation, the supernatant discarded, and the cells resuspended in0.1 M citrate buffer, pH 5.0. Cells were treated with 100 μg/mlnitrosoguanidine (NTG) for 25 min. at room temperature without shaking.The suspensions were centrifuged, the supernatant decontaminated, andthe cells were washed three times with 0.1 M potassium phosphate buffer,pH 7.0. Pellet was suspended in phosphate buffer and diluted into flaskscontaining 20 ml YM and shaken overnight. Cells were plated on YM andincubated at 20° C. Colonies were isolated on the basis of observedpigmentation differences, with the colonies characterized by darkreddish orange as observed under a dissecting microscope being selected,and streaked to slants. Three day old slants were used to inoculate YMflasks. One ml of YM medium was used to suspend the contents of theslant and this entire suspension was added to 250 ml flasks containing20 ml YM and shaken for 6 days at 20° C.

Morphological Selection

Example 3 Antimycin Treatment

Exponentially growing yeast cells of strain ATCC 24230, either NTGmutagenized as described under Step 1(a) above or non-mutagenized, wereplated on YM medium containing 50 μm antimycin A (a mixture ofantimycins A₁ and A₃; Sigma catalog #2006) and incubated at 20° C. untila colony grew. Growth took approximately one month. After two more weeksof incubation a red center appeared in the colony presenting a fried eggappearance. This red center was isolated to YM medium.

Example 4 Tunicamycin Treatment

Tunicamycin mutants were selected by plating a log phase culture ofstrain ATCC 24202 (naturally occurring) on a gradient plate of GYE agarwith 5 μg/ml tunicamycin. Colonies which grew on these plates werestreaked to GYE agar plates containing 5 and 8 μg/ml tunicamycin.Colonies which grew on these secondary plates were assayed for pigment.Control was 270 μg/g as compared to 610 μg/g for 8 μg/ml tunicamycinresistant mutant.

Example 5 Nystatin Treatment

Nystatin mutants were isolated by inoculating log phase YM cultures ofstrain ATCC 24202 into YM flasks containing 3 and 5 μg/ml nystatin.After 48 hours, these selective cultures were plated to antibioticplates containing YM agar with nystatin (3 and 5 μg/ml). Coloniesobtained were assayed for pigment. Control was 270 μg/g as compared to400 μg/g for highest nystatin mutant.

Example 6 Mevalonic Acid Treatment

Strain ATCC 24202 was mutagenized with NTG to 50% survival and plated toYM medium. Colonies were randomly plated to YM agar containing 5 mMmevalonic acid lactone. All of these colonies were subsequentlytransferred to medium containing first 10 and then 25 mM mevalonic acidlactone. Colonies which grew on 25 mM mevalonic acid lactone wereassayed for pigment. Control was 275 μg/g as compared to 540 μg/g for 25mM mevalonic acid lactone resistant mutant.

Carotenoid Extraction and Analysis

Colonies may be analyzed for pigment content as follows.

After harvest from 30 ml growth medium, the cells are washed in waterand resuspended in 30 ml of water. The optical density is measured todetermine growth. Approx. 13 ml of the suspension is broken with 0.5 mmglass beads for 3 to 4 minutes with cooling within a Bead Beater (BioSpec Products, Bartlesville, Okla.). After breakage, the bead/cellmixture is poured into a beaker and extracted 5× with 10 ml aliquots ofacetone. The acetone extracts are pooled and centrifuged at 15,000 rpmfor 45 min. The clear acetone supernatant is poured off the cell pellet;if the pellet contains residual pigment, it is manually ground with aglass homogenizer and further extracted with acetone.

The combined acetone extracts are combined in a separatory funnel, andapproximately 10 ml of petroleum ether is added as well as a few ml of asolution of saturate NaCl to help break the emulsion. The petrol extractis collected and the acetone layer is re-extracted. The petrol extractsare combined and filtered through glass wool to remove lipid globulesand other particulate matter. The absorbance spectrum is recorded (theabsorbance maximum of transastaxanthin in petroleum ether is 474 nm).The total carotenoid composition is calculated using the 1% extinctioncoefficient=2100 by the formula:

${{Total}\mspace{14mu}{carotenoid}\mspace{14mu}\left( {\mu\;{g/g}\mspace{14mu}{yeast}} \right)} = \frac{\left( {{ml}\mspace{14mu}{petrol}} \right)\mspace{14mu}\left( {{absorbance}\mspace{14mu} 474\mspace{14mu}{nm}} \right)\mspace{14mu}(100)}{(21)\mspace{14mu}\left( {{dry}\mspace{14mu}{weight}\mspace{14mu}{{yeast}\mspace{14mu}\lbrack g\rbrack}} \right)}$

Individual carotenoids may be analyzed by thin layer chromatography(TLC) and electronic absorption spectra. To prepare carotenoid extractsfor analysis, petroleum ether extracts are dried over anhydrous Na₂SO₄and concentrated by evaporation in a stream of nitrogen. These arechromatographed by TLC on silica gel plates (Silica gel 60, 5×20 dm,0.25 mm thickness (E. Merck, Darmstadt, West Germany) using 20%acetone/80% petroleum ether. After development, bands are scraped andeluted in acetone through a Pasteur pipet plugged with glass wool.Absorbance maxima, r_(f) values, and cochromatography with standards areused for identification of the pigments. Visible absorption spectra arerecorded in acetone or petrol on a Gilford Response spectrophotometer,and concentrations of carotenoids are calculated using the specificabsorption coefficients listed by Davies, Carotenoids, p. 38-165, inChemistry and Biochemistry of Plant Pigments, vol. 2., T. W. Goodwin(ed.), Academic Press, London (1976).

Bench Scale Fermentation Example 7

The production of astaxanthin in a 10 liter laboratory fermenter isillustrated by the following procedure:

20 ml. of frozen Phaffia rhodozyma strain IGI-887J1 (see FIG. 1 andaccompanying text) was used to inoculate 300 ml of a medium comprising3% glucose, 1% yeast extract and 0.1% caesin hydrolysate. The medium wassterilized at 121° C. and 15 p.s.i. for about 20 minutes before beinginoculated. The inoculated medium was shaken for ≈48 hours at atemperature of −20° C. on a rotary shaker with a 1 inch stroke at aspeed of 250 r.p.m. The 300 ml cell broth was used to inoculate a 5liter seed fermenter, containing 3 liters of sterilized seed mediumhaving the following composition per liter:

G Ammonium sulphate 5.0 Potassium phosphate monobasic 1.5 Magnesiumsulphate heptahydrate 1.5 Calcium chloride dihydrate 0.1 Yeast extract6.0 Vitamin mixture 2 ml Trace element mixture 1 mlThe vitamin mixture consists of the following ingredients:

G Biotin 0.08 Inositol 5.00 Thiamine 5.00 Calcium Pantothenate .00Pyroxidine HCL 2.25 Water, q.s. to make 1LThe trace element mixture consists of the following ingredients:

G 1) FeCl₃•gH₂O 3 2) Na₂MoO₄•2H₂O 2 3) ZnSO₄•7H₂O 8 MnSO₄•H₂O 3CuSO₄•5H₂O 6 Water, q.s. to make 1L

After inoculation, a 70% cerelose solution was fed incrementally to thefermenter, specific growth rate of the cells was kept at about u=0.15.The fermenter was maintained at a temperature of 20° C. pH wascontrolled at 5 with 8N KOH. Agitation was at the rate of 900 r.p.m. andaeration was at the rate of 1.5 v/v/m. When OD₆₆₀ (optical density) wasabout 30, 1 L of the broth was used to inoculate a 15 liter fermentercontaining 10 liter of production medium.

The composition of the production medium was the same as the seed mediumexcept: (1) ammonium sulphate was increased to 16 g/L, and (2) 1 g/Lmonosodium glutamate and 5 g/L corn steep liquor was added. Thefermenter temperature was maintained at 20° C. and pH was maintained at5 with 8N KOH. Agitation and aeration was at the rate of 900 r.p.m. and1.5 v/v/m respectively. About 2 liter of 70% cerelose was fedincrementally to the fermenter, specific growth rate of cells was keptbelow 0.15. At the end of the fermentation (about hrs), the cellscontained about 1100 μg/g astaxanthin.

Scale-Up Fermentation Example 8

The production of astaxanthin on a pilot plant scale is illustrated bythe following procedure.

20 ml of frozen P. rhodozyma strain IGI 188JB1 (see FIG. 1 andaccompanying text) was used to inoculate 300 ml of growth medium, thecomposition of the medium and culture conditions was the same asdescribed in Example 1. After 48 hours, the 300 ml cell broth was usedto inoculate the first seed fermenter, containing 3 liter of asterilized first seed medium having the following composition per liter:

G Ammonium sulphate 4.0 Potassium phosphate monobasic 1.5 Magnesiumsulphate heptahydrate 1.5 Calcium chloride dihydrate 0.1 Yeast extract4.0 Corn steep liquor 10.0  Vitamin mixture* 2 ml Trace element mixture*1 ml *same as in Example 1

90 g of sterilized cerelose was added aseptically to the fermenter afterinoculation. The fermenter was maintained at 21° C., pH was controlledat 5 with 8N KOH. Agitation was set at 900 r.p.m. and aeration was at1.5 v/v/m. When the initial cerelose was used up, a sterilized 70%cerelose solution was incrementally fed to the fermenter. The specificgrowth rate of the cells was controlled at about 0.1. When cell growthreached an O.D. of about 20, the 3 liter cell broth was used toinoculate a 30 liter fermenter containing 20 liter of sterilized 2ndstage seed medium.

The composition of 2nd stage seed medium was the same as the 1st stageseed medium. Culture conditions were also the same with the exception ofa slower, 300 r.p.m. agitation rate. When the initial cerelose wasexhausted, a sterilized 70% cerelose, 7% ammonium sulphate solution wasincrementally fed to the fermenter until an O.D. of about 30 wasreached. The specific growth rate of the cells was controlled at about0.1. The 2nd seed was used to inoculate a 250 liter fermenter containing170 liter of sterilized production medium.

Except for a higher concentration of corn steep liquor (12 g/L), thecomposition of the production medium was the same as the 1st stage seedmedium. Temperature was maintained at 21° C., pH was controlled at 5with 8N KOH. Aeration at 250 L/min for the first 15 hrs was increased to300 L/min. Agitation at 150 r.p.m. for the first 15 hrs was increased to200 r.p.m. A sterilized solution of 70% cerelose, 7% ammonium sulphatewas fed incrementally to the fermenter. Specific growth rate of thecells was controlled at about 0.1. When 30 kg of cerelose was fed to thefermenter, the cells were harvested. The cells contained about 1000 ug/gastaxanthin.

Enhancement of Astaxanthin Biosynthesis

It has also been found that when antimycin or another inhibitor of themain respiratory chain are added to Phaffia rhodozyma cells, and thecells are exposed to light, the astaxanthin content of the yeast isconsiderably enhanced. The underlying mechanism for this phenomena isnot understood, but it could be hypothesized that when the primaryrespiratory pathway is inhibited, light acts as one of the triggers of asecondary respiratory (oxidative) pathway, having a net effect ofconsiderably stimulating the production of astaxanthin. Thus, thepresent invention comprises processes for increasing the astaxanthin orother carotenoid content of yeast, comprising growing the yeast in thepresence of a metabolic pathway inhibitor while inducing a secondaryrespiratory pathway. The secondary respiratory pathway may be induced bysuch influences as light, certain environmental conditions such as thoseknown to cause stress, nutrients, etc. The present invention is,however, not limited by the above hypothesis.

It will be seen from the experiments discussed below that enhancedastaxanthin biosynthesis can be induced by the above mentionedcombination of respiratory chain inhibitor with initiation of thesecondary respiratory channel.

Example 9

P. rhodozyma strains used were the natural isolate UCD-FST-67-385 (Phaffet al., 1972; Miller et al., 1976), mutant Ant-1-4 used above, andstrain 18-13-6, an astaxanthin enhanced mutant obtained by ethylmethanesulfonate (EMS) mutagenesis procedures (isolated on YM agar). They weregrown in yeast extract/malt extract/peptone/glucose medium (YM medium,Difco Co., Detroit, Mich.) as previously described (An et al., 1989) ina temperature controlled incubator/shaker (Environ-Shaker Model 3597,Lab-Line Instruments, Inc., Melrose Park, Ill.). Growth was determinedby the optical density (660 nm) of a washed cell suspension; 1 mg drycell weight per ml corresponds to an O.D. of 1.35.

Light was supplied by two Sylvania 20 watt Coolwhite fluorescent tubesheld 20-40 cm from the flask media surfaces. Flasks were inoculated withapproximately 10²-10⁶ cells of actively growing yeast. For darkcontrols, flasks were wrapped in aluminum foil. When insoluble chemicalswere included in the media, they were first dissolved in a smallquantity of ethanol (0.3% final conc.), which did not affect yeastgrowth or pigmentation.

Carotenoid Extraction and Analysis

P. rhodozyma was grown for 5 days in flasks before extraction. Yeastswere harvested from liquid media by centrifugation. The yeast cells weresuspended in distilled water, washed in water, and extracted andanalyzed for carotenoids by thin-layer chromatography and absorptionspectroscopy as previously described (An et al., 1989).

Secondary Respiratory Pathway

Induction Increases Carotenoid Production

The influence of two 20 watt fluorescent bulbs placed 20 cm from thesurface of P. rhodozyma natural isolate, UCD-FST-67-385, and itsantimycin-sensitive mutants, Ant 1-4 and 18-13-6 was studied.

Separate cultures were grown under conditions of (a) total darkness for30 hours (hereafter “dark”), (b) total darkness with 0.2 μM antimycin(antimycin being introduced at inoculation) (hereafter“dark/antimycin”), and under conditions (c) and (d) which were identicalto (a) and (b) except for exposure of the cultures to light fromapproximately 30 hours into the experiment until approximately 60 hoursinto the experiment (hereafter “light” and “light/antimycin”).

Extraction of the pigments and characterization indicated that thepigments were qualitatively similar in composition, except that morecis-astaxanthin and lower carotene concentrations were found to bepresent in light grown cells.

Analysis of yeasts showed that the carotenoid content of light/antimycinP. rhodozyma, UCD-FST-67-385 increased by two-fold over any of the dark,dark/antimycin or light cultures of this natural isolate (Table 1).Table 2 shows that mutant 16-13-6, which is antimycin sensitive,produced two-fold increase in carotenoid content in antimycin/light overthe light culture.

TABLE 1 Growth Carotenoid Conditions (mg/ml) (μg/g y) Dark 4.4 520Dark + Ant 1.6 480 Light 3.3 440 Light + Ant 2.2 1000

TABLE 2 Antimycin Not added Added Light Carotenoid Content (μgcarotenoids/g yeast) White 540 1410 Blue 1050 1360 Red 960 1210 Dark 8301013

As can be seen from the above results, growth and carotenoid formationof natural and astaxanthin enhanced P. rhodozyma is clearly influencedby light. The natural isolate, UCD-FST-67-385, and itsantimycin-sensitive mutants ant-1-4 and 18-13-6, each grew better andhad increased pigmentation in the dark, but were differently affected bylight.

Phaffia as a Food and Pigment Source

The harvested cells can be disrupted or broken using mechanical orenzymatic means or a combination thereof and fed to salmonids, etc. aspart of an overall nutritious diet. In order to enhance the stability ofastaxanthin in the disrupted or broken yeast against heat and air, it ispreferred to provide a protective coating or matrix as conventionallyprovided in the food art, for example, a natural gum.

Examples wherein P. rhodozyma is used as a food supplement for rainbowtrout (Salmo gairdneri), American lobsters, Coturnix quail, and layinghens are discussed in a Thesis of co-inventor Johnson entitled“Astaxanthin Production by the Yeast Phaffia rhodozyma and its use as aPigment Source in Animal Feeding”, Masters Thesis, University ofCalifornia at Davis, (1976), a copy submitted herewith.

As a consequence of the higher astaxanthin content per unit of cells ona dry basis in the yeast attained according to the present invention,the amount of yeast employed to induce pigmentation can be substantiallyreduced.

Variations on the design or operation of the above illustrativeembodiments may be readily made to adapt the inventive process tovarious operational demands, all of which are within the scope andspirit of the present invention.

1. An astaxanthin mutant Phaffia rhodozyma producing more astaxanthinthan naturally occurring Phaffia rhodozyma, said mutant producing morethan 700 micrograms of astaxanthin per gram of dry yeast per six-dayculture in YM medium, wherein the amount of astaxanthin is determined bymeasuring the absorbance at 474 nanometers of a petroleum ether extractof Phaffia rhodozyma using a 1% (w/v) extinction coefficient in a onecentimeter cuvette of
 2100. 2. The mutant yeast of claim 1, wherein saidmutant yeast produces more than 900 μg of astaxanthin per gram of dryyeast.
 3. The mutant yeast of claim 2, wherein said mutant yeastproduces more than 1100 μg of astaxanthin per gram of dry yeast.
 4. Themutant yeast of claim 3, wherein said mutant yeast produces more than1400 μg of astaxanthin per gram of dry yeast.
 5. The mutant yeast ofclaim 4, wherein said mutant yeast produces more than 1700 μg ofastaxanthin per gram of dry yeast.
 6. The mutant yeast of claim 1,wherein said mutant yeast produces astaxanthin at a level at least twotimes that of naturally occurring Phaffia rhodozyma.
 7. The mutant yeastof claim 6, wherein said mutant yeast produces astaxanthin at a level atleast three times that of naturally occurring Phaffia rhodozyma.
 8. Themutant yeast of claim 7, wherein said mutant yeast produces astaxanthinat a level at least four times that of naturally occurring Phaffiarhodozyma.
 9. The mutant yeast of claim 8, wherein said mutant yeastproduces astaxanthin at a level at least five times that of naturallyoccurring Phaffia rhodozyma.
 10. The mutant yeast of claim 9, whereinsaid mutant yeast products astaxanthin at a level at least six timesthat of naturally occurring Phaffia rhodozyma.